HongTide can provide you high quality antibody production service to satisfy your research.
Some problems should be considered before we start our antibody progamme in order to make the antiboies more suitable for your research. Here are some of our suggestions:
Basic Requirements for Antibody Production
1. What kinds of experiments do you want to use for this antibody? (ELISA、IHC、Western Blot、IF or IP etc.)? (Functional requirements)
Scientists should establish proper functional requirements according to your experiments, because the antibody production difficulty will increase when the funtional requirements rise.
2. Are there commerical products for the antibody you need in the market? (Convenience requirements)
Commerical antibody should be the first choice if there are corresponding products in market becuase, it can save your time and quality of commerical products are usually more stable. But one antibody usually cannot satisfy the multi-functional requirements, so you need to choose more than one antibodies with different function to meet your experiment requirements.
3. What species of your antibody require (Rat, mice, goat, rabbit etc.)? (Species requirements)
Rabbit is usually used to produce polyclonal antibodies and for monoclonal antibodies, mice is the usual choice. We should use goat if the antibody amount requirements is large. Besides, kinship of antigen and immune host is another aspect we should consider, immunogenicity is better when the kinship is distant.
4. Monoclonal antibody or polyclonal antibody? (Character requirements)
Monoclonal antibody and polyclonal antibody have their own advantages respectively. Specificity of monoclonal antibody is better and the properties is more stable, we should choose monoclonal antibody when we need to identify antigens with higher homology or specificity. The advantage of polyclonal antibody is it has higher sensitivity and titer.
5. What do you want to use as antigen (CDS sequence, recombinant plasmid, recombinant protein, peptide etc.)? (Possibility requirements）
A good antigen is necessary for antibody production. You can immune animal directly if you already have proper antigen, but if not, you should try to prepare antigen from the informations or materials you have had: sequence information-gene-recombinant expression-protein purification- animal immunization. Carrier conjugated peptide is another way to get antigen if the protein has high homology.
6. Screning and test method (positive standard and antibody experiments confirming method)? (Testability requirements)
Screning and purification method is vital in antibody production, and test method control the fate of the antibody. A stable and reliable screening and purification method is necessary for antibody production.
7. Do you have experience or platform for antibody production? (Feasibility requirements)
Experienced staffs and professional platform are necessary for antibody production, if you don't have such experience and don't plan to build this kind of platform, entrusting the antibody to professional company is a good choice.
Basic Principle for Antibody Production
1. Appropriate amount
We should choose the production amount according to the experiment requirement and the difficulty of the antibody production. Usually, we need 200-400ug antigen to immune mice and at least 2mg for one rabbit. For our research, we should choose small animals such as rat if the antibody production is enough. Due to the individual difference, the antigen immuning one rabbit can immune 4-5 rat, which can avoid the time wasted by the animal individual difference and accident death.
2. Time principle
Time principle is mainly based on the situation when we need the antibody urgently. We should try to use fast anitbody production if the immunogenicity and purity of antigen is good enough. At present, many companies has fast antibody preparation service based on hydrosoluble fast adjuvant.
Primary Requirements for Animal Antigens
1. Large molecular weight
Molecules with large molecular weight usually have longer half-time in vivo, which make them has more oppotunities to interact with immunocytes such as macrophage, B lymphocytes and T lymphocytes and make immunocytes have immnue reponse. Contrast to large molecules, small molecules will be excreted to
the body and have little chance to interact with immunocytes and induce no immune reponse. Similarly, if large molecules are easily to be degarded and excreted rapidly, it is not a suitable substance as antigen. Normally, molecules < 4kD is hard to induce immune reponse.
2. Strong exogenous
At the early stages in the ontogenesis, bodies form immunologic tolerance to its own substance, substances similar to own substance are hard to induce immune response. Only when the exogenous substance invade the body, immune response would be induced. But substances from brain, eyes and testis which have its own natural barrier will not induce immune response. We can ignore the immunotolerance and exogenous if we want to use substances from them as antigens.
3. Structural complexity
Exogenous decscribe antigens from macro classification, but from the micro perspective, structures of antigens should be as complex as possible. Simply repeated sequence such as gelatin, starch, poly amino acids which are composed with surgars, amino acids usually have very weak antigenicity.
4. Easily to be degraded
Antigens which can easily be degraded can induce antigen presentation. Substances such as plastic and steel which are hard to be degraded have very weak immunogenicity. Substances composed with D-amino acids cannot be degraded, so they have weak immunogenicity too.
Usual Types and Preparation Methods of Antigens
1. Natural antigens
Natural proteins which are extracted from biological organisms retaining its own structures are very good antigens. But the purification for proteins is difficult and only the proteins with high expression abundance and stable properties can be purified. Antibodies produced by the antigens of natural proteins are usually used for ELISA, immunochromatography, Immunoturbidimetric diagnosis etc.
2. Recombinant Protein Antigens
There are three commonly used expression systems for the production of recombinant protein antigens which are prokaryotic expression system, yeast expression system and eukaryotic expression system. Usually, a tag such as GST, 6*His, Myc, MBP, Flag, Fc etc. will be attached to the protein when we use recombinant protein expression to produce antigens, which will enhance the solvability and M.W. of the antigen. Tag can be cleaved if needed. If the tags are not cleaved and the tag antibodies make disturbance to experiments, the tag antibodies can be removed using tag protein immunoadsorption. Comparing with natural protein, there are still difference between natural protein and recombinant protein. Protein expressed by eukaryotic expression system is more closely to natural proteion than prokaryotic expression system, which is usually used for the production of Western Blot, IHC, ELISA antibodies.
3. Peptide Antigens
Peptide antigens can be chemically synthesized, and complete antigens will be got when the peptides are conjugated to carriers such as BSA, RSA, HSA, OVA, GST, KLH, MAPs etc. Comparing with peptide synthesis, conjugating peptide to carriers is much more easier. Carboxyl group and amino group on peptide sequence can be used to conjugate peptides to carriers through EDC/NHS or EDC. If we want to conjugate peptide to carriers at fixed position, -SH can be added to peptides through adding a Cys to peptide, and conjugate -SH to carriers using Sulfo-SMCC.
Comparing with natural antigens and recombinant protein antigens, advantage of the peptide antigens is its enriched dominant epitopes, which can stimulate immune response. We usually use peptide antigens when the protein antigens is difficult to be extracted or expressed, or the protein is highly homologous with the proteins in biological organisms.
The difficulty of peptide antigen is the design and screen of peptide sequence(epitopes), with our professional computational biology team and database, we can provide you fast and accurate epitopes screening service.
4. Small Molecule Antigens
Aromatic small molecule can induce immune response. Similar with peptide, small molecules can form complete antigens only when they are conjugated to carrier proteins. Linkers are usually used to conjugate small molecules to carrier proteins.
5. Metal Ion Antigens
Metal ion antigens is achieved by chelates such as DOTA, NOTA etc. We usually conjugate chelates to protein carriers, then chelate metal ions to the carrier.
6. Tissues, Whole Cells or Cell Components Antigens
Tissues, whole cells or cell components antigens are usually used to produce HBsAg or cell antibodies. Cells must be seprated and the serum and cell fragments of cells must be removed to improve the purity of the cell when we use cells as antigens. The advantages of cell antigens are: the cells have complete granularity and exogenous, which can induce immnue reponse much more easier, conformation of the antibodies are more closely to the real structures. Cell antigens are usually injected to cavum abdominis with the dose more than 1.0×105 to induce the immnue response.
Hypotonic solution (low concentration buffers with MgCl2, MgCl2 is used to provent the broken of caryon) is usually used to extract cytomembrane and cytoplasm when we want to use them as antigens. We can use Ca2+ homogenate centrifugation if we only want to extract cytomembrane.
7. Whole Virus Particle Antigens
Devitalization of virus is needed when we want to use virus as antigens. The purity of virus should be as high as possible.
8. Bacterial Particle Antigens
Whole bacterial hyperimmune serum is usually applied to bacterio-agglutination immune experiments. Bacterium used as antigens shoulb be in good shapes and conditions. Devitalization of bacterium is needed too.
Matters Needing Attention
1. Purity
The higher of and antigen purity is, the easier of the postprocessing will be. For polyclonal antibodies, we can get high titer and low background antibodies if the antibody molecules aimed to antigens in hyperimmune serum we used is more. For monoclonal antibodies, we can get antibody secretory hybirdoma cell strains with high specificity and titer more easier if the antigen purity is higher.
2. Toxicity
Toxicity of antigens should be as low as possible, high toxicity will cause the death of animals, and cannot product high quality antibodies.
3. Solvent of Antigens
We should use PBS or physiological saline as solvent of antigens. If we cannot dissolve antigens using PBS or physiological saline, we can try low concentration urea because animal can bear low concentration urea.
4. Adjuvant
Choosing a proper adjuvant is another important step in antibody production. Freund's adjuvant, developed by Freund, is the major adjuvants at present. Main component of Freund's adjuvant is mineral oil and sugar. There are two types Freund's adjuvant, which are Freund's complete adjuvant and Freund's incomplete adjuvant. Freund's complete adjuvant is Freund's incomplete adjuvant added with BCG. Function of adjuvant is to extend release of hydrosoluble antigens.
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